Method of treating biological materials with translating electrical fields and electrode polarity reversal

ABSTRACT

A method and apparatus are provided for treating biological cellular material with a treating agent using pulsed electrical fields provided by a waveform generator ( 12 ). The treatment method includes obtaining an electrode assembly which includes three or more parallel rows of individual electrodes ( 19 ). The electrode assembly is applied to a treatment area. Electrically conductive pathways are established between the electrodes ( 19 ) and the waveform generator ( 12 ) through an array switch ( 14 ). Successive electric fields are applied to the treatment area in the form of successive electric field waveforms from the waveform generator ( 12 ), through the array switch ( 14 ), to adjacent rows of electrodes ( 19 ), wherein each successive electric field has the same direction, and wherein polarities of rows of electrodes are reversed successively during the applying of the successive electric fields between adjacent successive rows of electrodes to the treatment area. As a result, the biological cellular material in the treatment area is treated with the treating agent unidirectionally with uniform electric fields with a minimization of the formation of deleterious electrochemistry products at the electrodes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to pending U.S. provisional patentapplication for METHOD OF TREATING BIOLOGICAL MATERIALS WITH TRANSLATINGELECTRICAL FIELDS AND ELECTRODE POLARITY REVERSAL, Ser. No. 60/372,436,Filing Date 16 Apr. 2002. This application is also related to pendingU.S. patent application for ELECTRODES COATED WITH TREATING AGENT ANDUSES THEREOF of King and Walters, Ser. No. 09/920,861, Filing Date 03Aug. 2001, which is related to copending PCT International ApplicationNumber PCT/US00/00014, filed 12 Jan. 2000, which is based upon copendingU.S. Provisional Application Ser. No. 60/117,755, filed 28 Jan. 1999,and which was published on 3 Aug. 2000 with PCT InternationalPublication Number WO 00/44438.

TECHNICAL FIELD

This invention relates to the method of delivering therapeutic materialsinto living cells using pulsed electric fields. More specifically, thepresent invention provides methods and apparatus for deliveringsubstances, such as macromolecules and chemotherapeutic agents intocells, in vivo, ex vivo, in vitro, and in tissues.

BACKGROUND ART

Electroporation is the reversible destabilization of cell membranes byapplication of a brief electric field across the cell resulting in apotential across the cell membrane. Properly administered, thedestabilization results in a temporary pore or pathway through whichtherapeutic material can pass. The uses of electroporation are many.Some are: (1) transient introduction of DNA or RNA, (2) permanenttransfection of DNA, (3) introduction of antibodies, or other proteinsor drugs into cells, (4) gene therapy, and (5) cancer vaccinations, etc.

To deliver the therapeutic compounds into living cells usingelectroporation, a system consisting of three components is required:(1) a pulse voltage waveform generator, (2) a switching device toconnect the anode or cathode of the pulse voltage waveform generator tothe electrodes, and (3) an electrode array to convert the pulse voltageinto a pulsed electric field. The electrode can designed for in vitrodelivery in an aqueous solution or for in vivo delivery into tissue.

In the most elementary system, where the array of electrodes consists ofa single anode and a single cathode, the switching device is notrequired. The primary objective of the electrode array is to provide auniform electric field over the area of cell treatment.

A number of patent, published, applications, and literature referencesare relevant to these matters, and they include the following:

U.S. Pat. No. 5,674,267, issued Oct. 7, 1997, of Mir et al.

U.S. Pat. No. 5,702,359, issued Dec. 30, 1997, of Hofmann

U.S. Pat. No. 5,873,849, issued Feb. 23, 1999, of Bernard

U.S. Pat. No. 5,993,434, issued Nov. 30, 1999, of Dev et al.

U.S. Pat. No. 6,010,613, issued Jan. 4, 2000, of Walters et al.

U.S. Pat. No. 6,014,584, issued Jan. 11, 2000, of Hofmann et al.

U.S. Pat. No. 6,055,453, issued Apr. 25, 2000, of Hofmann et al.

U.S. Pat. No. 6,110,161, issued Aug. 29, 2000, of Mathiesen et al.

U.S. Pat. No. 6,117,660, issued Sep. 12, 2000; of Walters et al.

International Patent Publications

PCT/GB01/00899, published 02 Mar. 2, 2001, of Shirkhanzadeh

PCT/US00/00014, published 12 Jan. 2000, of King et al.

Literature Publications

-   Hofmann et al., “Electrochemotherapy: Transition from Laboratory to    the Clinic”, IEEE Engineering in Medicine and Biology.,    November/December 1996.-   Mir et al., “High-efficiency gene transfer into skeletal muscle    mediated by electric pulses”, Proc. National Academy of Sciences,    USA, Vol. 96, pp 4262-4267, April 1999.-   Gehl, et al., “In vivo electroporation of skeletal muscle:    threshold, efficacy and relation to electric field distribution”,    Biochimica et Biophysica Acta 1428 (1999) 233-240.-   Loomis-Husselbee, J. W., Cullen, P. J., Irvine, R. F., &    Dawson, A. P. (1991). Electroporation can cause artefacts due to    solubilization of cations from the electrode plates. Aluminum ions    enhance conversion of inositol 1,3,4,5-tetrakisphosphate into    inositol 1,4,5-trisphosphate in electroporated L1210 cells. Biochem.    J, 277, 883-885-   Friedrich, U., Stachowicz, N., Simm, A., Fuhr, G., Lucas, K.,    Zimmermann, U. High efficiency electrotransfection with aluminum    electrodes using microsecond pulses-   Stapulionis, R. (1999). Electric pulse induced precipitation of    biological macromolecules in electroporation. Bioelectrochem.    Bioenerg., 48, 249-254-   Tomov, T. & Tsoneva, I. (2000), Bioelectrochemistry., 51, 207-209-   Kotnik, T., Miklavcic, D., Mir, L. M. Cell membrane    electropermeabilization by symmetrical bipolar rectangular pulses,    Part II. Reduced electrolytic contamination-   Bockris, J. O., Reddy; A. K. N. editors; Modern electrochemistry.    Plenum/Rosetta, 1977

In Mir et al, (U.S. Pat. No. 5,674,267), an array of concentric needlesis suggested. In this array two needles, one anode and one cathode areselected from all of the needles of the array. A switching device isused to select many pairs to cover the treatment area. One pair ofneedles has coverage of only a limited area. Moreover, even by selectingall needle pairs in the area, it is not possible to provide uniformcoverage of the total treatment area. In this respect, it would bedesirable to provide an electrode array and a method for selectingelectrodes in the electrode array that would provide uniform coverage oftotal treatment area.

Hofmann (U.S. Pat. No. 5,702,359) and Dev (U.S. Pat. No. 5,993,434)disclose similar systems wherein two anodes and two opposing cathodesare selected at a time. That, is two pairs of opposing anodes andcathodes are selected at a time. There are a total of six pairselectrodes, of which two pairs at a time would be connected to the pulsegenerator by a switching device. More recently, Hofmann et al (U.S. Pat.Nos. 6,014,584 and 6,055,453) disclose the use of an array of needleelectrodes and connecting two opposing pairs of electrodes at a time,and rotating those pairs 90 degrees.

Bernard (U.S. Pat. No. 5,873,849) discloses an electrode systemconsisting of rows of offset needle electrodes in which at least threeelectrodes are arrayed in an equilateral triangle, and these electrodesare connected to the pulse generator. This array consists of one or moreequilateral triangles with two electrodes connected to one polarity ofthe pulse generator and the third electrode connected to the oppositepolarity. In this system, various electrodes could be connected to treatthe coverage area. The combination of one electrode with one polarityand two electrodes of the opposite polarity leave significant areas fortreatment where the electric field is not effective. In this system, twoelectric field vectors point in different directions.

In 1999, Gehl et al published the above-mentioned paper in which theydisclose an array of two parallel rows of needle electrodes. Thiselectrode array provides a very uniform electric field within the rows,and the electric field provided is almost as uniform as an electricfield provided between two parallel plates. However, as the treatmentarea increases the distance between the rows of needles increases. Asthe distance between the rows of needles increases, the uniformity ofthe field decreases, and the voltage to maintain the sale electric fieldstrength must increase.

The pulse waveforms of the switching systems in the patents andpublications described thus far above were generally rectangular waves,having the same pulse width and interval, and employing a few pulses.

Then, Walters et al, in U.S. Pat. No. 6,010,613, which is incorporatedherein by reference, introduced waveforms, whose pulse parameters can bechanged on a pulse to pulse basis. These waveforms are used to formpores using short duration electric field pulses (in the microsecondrange) and then to move large charged molecules into the cells with aseries of lower and longer duration (in the milliseconds range) electricfield pulses.

Mir et al. then published, in Proc. National Academy of Science, USA,April 1999 cited above, disclosing that electric field duration of 10'sof milliseconds was optimum for the transfection of skeletal muscle.These longer pulse, widths produced a problem—that of electrochemistryeffects in the vicinity of the electrodes.

Shirkhanzadeh, in the PCT/GB01/00899 publication mentioned above,addressed the electrode electrochemistry effects resulting from thelonger pulses by using palladium electrodes one of which was infusedwith hydrogen.

Using bipolar pulses can also minimize electrochemistry at electrodes.Mathiesen (U.S. Pat. No. 6,110,161) discloses using bipolar pulses oflow electric field strength and moderate duration (50 to 5000microseconds) for in vivo electroporation of skeletal muscle, but didnot specifically address the electrochemistry effects.

In one respect, using bipolar pulses may address the electochemistryproblems, but, in another respect, the use of bipolar pulses is counterproductive. The first pulse will move larger charged molecules, in onedirection and a second pulse immediately following of opposite polaritythen moves the large charged molecules back. A method is needed to keepmoving the large charged molecule in the same direction to improvedelivery into living cells while simultaneously minimizing theelectrochemistry effects.

In methods involving electroporation of cells described above, studieshave been made relating to the local environments at the electrodes andon the surfaces of the electrodes. In this respect, studies haverevealed that metal ions are released by such electrodes.

In Loomis-Husselbee, J. W., Cullen, P. J., Irvine, R. F:., & Dawson, A.P. (1991). Electroporation can cause artefacts due to solubilization ofcations from the electrode plates. Aluminum ions enhance conversion ofinositol 1,3,4,5-tetrakisphosphate into inositol 1,4,5-trisphosphate inelectroporated L1210 cells. Biochem. J, 277, 883-885, Loomis-Husselbeeet al demonstrated that aluminum ions are generated by aluminumelectrodes during electroporation. The aluminum ions inhibited thebiochemical process under investigation. The authors concluded thataluminum ions produced during electroporation can be detrimental tocells.

In Friedrich, U., Stachowicz, N., Simm, A., Fuhr, G., Lucas, K.,Zimmermann, U. High, efficiency electrotransfection with aluminumelectrodes using microsecond pulses Friedrich et al showed thatsubstantial amounts of aluminum were released from aluminum electrodesduring long pulses. The principle cause of the aluminum ion release wasa change of pH at the electrode interface produced by electrolysis ofwater. Aluminum ion release was reduced when short pulses were used.

In Stapulionis, R. (1999). Electric pulse-induced precipitation ofbiological macromolecules in electroporation. Bioelectrochem. Bioenerg.,48, 249-254, Stapulionis et al showed that aluminum, iron, or copperions were produced by the anode of metal electrodes duringelectroporation. The metal ions produced by this process precipitatedmacromolecules. The macromolecule precipitation resulted in reducedreagent and reduced delivery of macromolecules into cells.

In Tomov, T. & Tsoneva, I. (2000), Bioelectrochemistry, 51, 207-209,Tomov et al observed that metal ions are produced by stainless steelelectrodes during electroporation similar to the release of aluminumions from aluminum-containing electrodes. More iron was released byhigher electric fields, wider pulse widths and increased saltconcentration. The potential for harmful effects of iron were discussed.Quantitatively less iron is released from stainless steel electrodesthan is released from aluminum electrodes (shown by others).

In Kotnik, T., Miklavcic, D., Mir, L. M. Cell membraneelectropermeabilization by symmetrical bipolar rectangular pulses, PartII. Reduced electrolytic contamination, Kotnik et al compared therelease of aluminum from aluminum electrodes and iron from stainlesssteel electrodes. The effects of unipolar and bipolar pulses on metalion release induced by the two types of metal electrodes were compared.As was seen by others, significant amounts of metal ions were releasedwhen unipolar pulses were used for electroporation. There was asignificant reduction in metal ion production when bipolar pulses wereused.

There is a general discussion of events occurring at interfaces of metalconductors and ionic conductors during electroporation in Bockris, J.O., Reddy, A. K. N. editors, Modern electrochemistry. Plenum/Rosetta,1977. This discussion reveals that electrodes used for in vivo or invitro electroporation are electronic conductors. The tissue or fluidsurrounding the electrode is an ionic conductor. The interface betweenthe two is complicated. Electrolysis occurs at the electrodes. At rest,there are ion clouds in the ionic conductor at the interface that inducea charge equal in strength and opposite in charge within the electronicconductor. When an electrical potential is applied across at least twooppositely charged electrodes (the electronic conductors) a current isinduced across the ionic conductor. The ionic current differs from thatin an electronic conductor in that ions are actively involved in thetransport of electrons through the solution. The current is aunidirectional flow of electrons through the solution by ionicconductance.

One electrode serves as a source of electrons (cathode) and anotherserves as a sink for uptake of electrons (anode). At the electron sourceions are electronated or reduced. At the electron sink, ions arede-electronated or oxidized. As an example, hydrogen ions areelectronated and form hydrogen molecules at the electronating electrode.At the electron sink electrode, oxygen is formed by de electronation ofwater. Other ions can undergo the same process.

Many of the products of electrolysis are detrimental to theelectroporation process. They interfere with the electrode-ionicconductor interface and they can be toxic to cells. In addition, metalsfrom the electrode can be introduced into the solution by electrolysisor corrosion.

Bipolar pulses reverse the polarity of the electrodes. This causes theelectronation electrode to become the de-electronation electrode and thede-electronation electrode to become the electronation electrode. Thisreversal causes a reversal of electrochemistry effects and thus reducesthe negative effects of unipolar pulses.

From a study of the prior art, and from the present inventorsdiscoveries, a number of conclusions have been derived relating toelectrical pulses, electroporation, deleterious electrode effects, andcell uptake of treating agents. First, although unipolar pulses canprovide an electroporation environment in which good macromoleculeelectrophoresis and good levels of cell uptake of treating agents areobtained, deleterious electrode effects are a problem. Second, and incontradistinction with the first, although bipolar pulses can provide anelectroporation environment in which deleterious electrode effects arenot a problem, poor macromolecule electrophoresis and poor levels ofcell uptake of treating agents are obtained. In this respect, it wouldbe desirable if an electroporation method were provided in which thebenefits of using unipolar pulses were obtained without incurring thedisadvantages of unipolar pulses, and in which the benefits of usingbipolar pulses were obtained without incurring the disadvantages of thebipolar pulses.

More specifically, it would be desirable to provide a method of treatingbiological materials with electrical fields and treating agents whichemploys unipolar pulses but which has minimal deleterious electrolyticeffects at the electrodes.

Also, it, would be desirable to provide a method of treating biologicalmaterials with electrical fields and treating agents which employsunipolar pulses and retains good electrophoresis properties for goodcell uptake of treating agents.

Also, it would be desirable to provide a method of treating biologicalmaterials with electrical fields and treating agents which employsunipolar pulses but which also employs electrode polarity reversal.

Also, it would be desirable to provide a method treating biologicalmaterials with electrical fields and treating agents which employselectrode polarity reversal without employing bipolar pulses.

Thus, while the foregoing body of prior art indicates it to be wellknown to use electroporation and electrophoresis for driving treatingagents into cells, the prior art described above does not teach orsuggest a method of delivering therapeutic treating agents into livingbiological cells, especially living mammalian cells, which has thefollowing combination of desirable features: (1) can produce a pulsedelectric field over the treatment volume that is uniform andunidirectional;

(2) can be scaled to produce the same uniform and unidirectionalelectric field over larger or smaller treatment volumes with the sameapplied pulse voltage;

(3) can produce, without removing the electrode array a second uniformand unidirectional electric field over the treatment volume at 90degrees or 180 degrees or 270 degrees with respect to the direction ofthe first electric field;

(4) can produce, without removing the electrode array, a third or fourthuniform and unidirectional electric field over, the, treatment volumethat are 90 degrees or 180 degrees or 270 degrees with respect to thedirection of the first electric field;

(5) can minimize adverse electrochemistry activity at the electrodes,without using bipolar electric fields, when pulse widths that are longerthan a few hundred microseconds are used;

(6) can reduce heating in the treatment volume by applying the electricfield sequentially to adjacent segments of the treatment volume;

(7) provides an electroporation method in which the benefits of usingunipolar pulses are obtained without incurring the disadvantages ofunipolar pulses;

(8) provides an electroporation method in which the benefits of usingbipolar pulses are obtained without incurring the disadvantages of thebipolar pulses;

(9) provides a method of treating biological materials with electricalfields and treating agents which employs unipolar pulses but which hasminimal deleterious electrolytic effects at the electrodes;

(10) provides a method of treating biological materials with electricalfields and treating agents which employs unipolar pulses and retainsgood electrophoresis properties for good cell uptake of treating agents;

(11) provides a method of treating biological materials with electricalfields and treating agents which employs unipolar pulses, but which alsoemploys electrode polarity reversal; and

(12) provides a method of treating biological materials with electricalfields and treating agents which employs electrode polarity reversalwithout employing bipolar pulses.

The foregoing desired characteristics are provided by the unique methodof the invention of treating biological materials with translatingelectrical fields and electrode polarity reversal for driving treatingagents into the biological materials. More aspects of the presentinvention as will be made apparent from the following descriptionthereof. Other advantages of the present invention over the prior artalso will be rendered evident.

DISCLOSURE OF INVENTION

It is noted that this application is related to pending U.S. provisionalpatent application for METHOD OF TREATING BIOLOGICAL MATERIALS WITHTRANSLATING ELECTRICAL FIELDS AND ELECTRODE POLARITY REVERSAL, Ser. No.60/372,436, Filing Date 16 Apr. 2002. In addition aspects of theinvention have been disclosed in pending U.S. patent application forELECTRODES COATED WITH TREATING AGENT AND USES THEREOF of King andWalters, Ser. No. 09/920,861, Filing Date 03 Aug. 2001, and in copendingPCT International Application Number PCT/US00/00014, filed 12 Jan. 2000,which is based upon copending U.S. Provisional Application Ser. No.60/117,755, filed 28 Jan. 1999. The PCT International Application No.PCT/US00/00014 was published on 3 Aug. 2000 with PCT InternationalPublication No. WO 00/44438, which is incorporated herein by reference.In addition to currently disclosing some of those aspects of theinvention previously disclosed in the above-mentioned PCT and theabove-mentioned U.S. Provisional Application Ser. No. 60/117,755, filed28 Jan. 1999, the present application discloses additional inventionaspects.

This application relates to treating biological cells. The biologicalcells can be in vivo, ex vivo, or in vitro. More, specifically thebiological cells can be in epidermal tissue and can be Langerhans cellsin the epidermal tissue. Also, the biological cells can be deep tissues,and can be in tumors in deep tissues.

The principles of the present invention can be stated in a number ofways.

In accordance with one aspect of the present invention, a method oftreating material with a treating agent is provided using pulsedelectrical fields provided by a waveform generator. The method includesthe steps of:

obtaining an electrode assembly which includes three or more parallelrows of individual electrodes,

establishing electrically conductive pathways between the electrodes andthe waveform generator, and

applying successive electric fields in the form of successive electricfield waveforms from the waveform generator to adjacent rows ofelectrodes, wherein each successive electric field has the samedirection, and wherein polarities of rows of electrodes are reversedsuccessively during the applying of the successive electric fieldsbetween adjacent successive rows of electrodes.

In accordance with another aspect of the present invention, a method oftreating material with an agent is provided using pulsed electricalfields provided by a waveform generator. The method includes the stepsof:

a. obtaining an electrode assembly which includes K rows of electrodes,where K is at least three, wherein each successive row of electrodes isspaced apart from a preceding row of electrodes,

b. establishing electrically conductive pathways between the K rows ofelectrodes and the waveform generator, and

c. providing successive electric fields in the form of successiveelectric field waveforms from the waveform generator to the K rows ofelectrodes, wherein each electric field has the same direction,

(a) such that an Lth electric field is applied between a selected Lthrow of electrodes and an (L+1)th row of electrodes among the K rows ofelectrodes, wherein L+2 is less than or equal to K, wherein the Lth rowof electrodes has a first polarity, and the (L+1)th row of electrodeshas a second polarity, and

(b) such that, subsequently, an (L+1)th electric field is appliedbetween the (L+1)th row of electrodes and an (L+2)th row of electrodes,wherein the (L+1)th row of electrodes has the first polarity, and the(L+2)th row of electrodes has the second polarity, and

d. repeating step c. as many times as desired with as many selections ofL as desired, such that L+2 is less than or equal to K.

Each of the K rows of electrodes can include at least three individualelectrodes.

The electric field waveforms can be pulsed electric field waveforms.

The electric field waveforms can be unipolar electric field waveforms.

The pulsed electric field waveforms can be from rectangular pulses.

The pulsed electric field waveforms can be from electrical pulses whichare in a sequence of at least three non-sinusoidal electrical pulses,has field strengths equal to or greater than 100 V/cm, to the material,wherein the sequence of at least three non-sinusoidal electrical pulseshas one, two or three of the following characteristics (1) at least twoof the at least three pulses differ from each other in pulse amplitude,(2) at least two of the at least three pulses differ from each other inpulse width, and (3) a first pulse interval for a first set of two ofthe at least three pulses is different from a second pulse interval fora second set of two of the at least three pulses.

The first polarity can be positive, and the second polarity can benegative. Alternatively, the first polarity can be negative, and thesecond polarity can be positive.

Successive electric fields can be applied unidirectionally from thefirst and second rows of electrodes to the Kth row of electrodes. Then,successive electrical fields can be applied unidirectionally from theKth row of electrodes and: (K−1)th row of electrodes to the first row ofelectrodes, which is in reverse direction.

The material treated can be biological material The biological materialcan be cellular material. The cellular material can be skin cells,tissue, deep organ tissue, muscle tissue, and mammalian cells, amongothers.

The treating agent can includes molecules of electrode releasable tissuetreating agent on the electrodes, which are released from the electrodesby applying electrophoretic pulses to the electrodes. The molecules ofthe electrode releasable tissue treating agent can be released from theelectrodes by contacting the electrodes with a solvent.

In accordance with another aspect of the present invention, a method forimmunotherapy is provided which includes the steps of:

a. obtaining an electrode assembly which includes K rows of electrodes,where K is at least three, wherein each successive row of electrodes isspaced apart from preceding row of electrodes, wherein each electrode isstatically-coated with an immuno-stimulating material,

b. establishing electrically conductive pathways between the K rows ofelectrodes and a waveform generator,

c. inserting the statically-coated electrodes into a tissue to betreated,

d. releasing the immuno-stimulating material from the electrodes,

e. providing successive electric fields in the form of successiveelectric field waveforms from the waveform generator to the K rows ofelectrodes, such that the released immuno-stimulating material is driveninto cells in the tissue, wherein each electric field has the samedirection,

-   -   (a) such that an Lth electric field is applied between a        selected Lth row of electrodes and an (L+1)th row of electrodes        among the K rows of electrodes, wherein L+2 is less than or        equal to K. wherein the Lth row of electrodes has a first        polarity, and the (L+1)th row of electrodes has a second        polarity, and    -   (b) such that, subsequently, an (L+1)th electric field is        applied between the (L+1)th row of electrodes and an (L+2)th row        of electrodes, wherein the (L+1)th row of electrodes has the        first polarity, and the (L+2)th row of electrodes has the second        polarity, and

f. repeating step e. as many times as desired with as many selections ofL as desired, such that L+2 is less than or equal to K.

The molecules in the static coating can be a solid phase, a gel, andmacromolecules such as a polynucleotide vaccine, a solid phasepolynucleotide vaccine, a DNA vaccine, a solid phase DNA vaccine, an RNAvaccine, a solid phase RNA vaccine, a protein-based vaccine, a solidphase protein-based vaccine, an organ treating agent, and a deep tissuetumor treating agent, among others.

The immuno-stimulating material can be released from the electrodes byapplying electrophoretic pulses to the electrodes. Alternatively, theimmuno-stimulating material can be released from the electrodes bycontacting the electrodes with a solvent. The immuno-stimulatingmaterial can be released from the electrodes by contacting theelectrodes with a solvent which includes body fluids.

The electrode assembly can include a plurality of electrodes arranged inat least three parallel rows of electrodes. The at least three parallelrows of electrodes can include at least three parallel plate electrodes.

The parallel rows of electrodes can include needle electrodes. Theneedle electrodes can include relatively short needles that penetrateskin only. The needle electrodes can include relatively long needlesthat penetrate tissues below the skin. The parallel rows of electrodescan include pad electrodes.

In accordance with another aspect of the present invention, a method oftreating material is provided using pulsed electrical fields provided bya waveform generator. The method includes the steps of:

obtaining an electrode assembly which includes a first electrode, asecond electrode spaced apart from the first electrode, and a thirdelectrode spaced apart from the second electrode,

establishing electrically conductive pathways between the electrodes andthe waveform generator,

locating the electrodes such that the material to be treated is situatedtherebetween, and

providing successive electric fields in a common direction in the formof successive pulse waveforms from the waveform generator applied to thematerial to be treated in the common direction, such that a firstelectric field is applied between the first electrode and the secondelectrode, wherein the first electrode has a first polarity, and thesecond electrode has a second polarity, and such that a second electricfield is applied between the second electrode and the third electrode,wherein the second electrode has the first polarity, and the thirdelectrode has the second polarity, wherein the first electric field andthe second electric field are in a common straight line direction.

The electrode assembly can further include a fourth electrode which isspaced apart from the third electrode, and which is located in thematerial to be treated, further providing an additional electric fieldin the form of an additional pulse waveform from the waveform generatorapplied to the material to be treated, such that a third electric fieldis applied between the third electrode and the fourth electrode. Thethird electrode has the first polarity, and the fourth electrode has thesecond polarity. The first, second, and third electric fields are in acommon straight line direction.

The electrode assembly can further include a fifth electrode which isspaced apart from the fourth electrode, and which is located in thematerial to be treated, further providing an additional electric fieldin the form of an additional pulse waveform from the waveform generatorapplied to the material to be treated, such that a fourth electric fieldis applied between the fourth electrode and the fifth electrode, whereinfourth electrode has the first polarity, and the fifth electrode has thesecond polarity. The first, second, third, and fourth electric fieldsare in a common straight line direction.

In accordance with another aspect of the present invention, a method ofproviding pulsed electrical fields provided by a waveform generatorincludes the steps of:

a. obtaining an electrode assembly which includes K rows of electrodes,where K is at least three, wherein each successive row of electrodes isspaced apart from a preceding row of electrodes,

b. establishing electrically conductive pathways between the K rows ofelectrodes and the waveform generator, and

c. providing successive electric fields in the form of successiveelectric field waveforms from the waveform generator to the K rows ofelectrodes, wherein each electric field has the same direction,

(a) such that an Lth electric field is applied between a selected Lthrow of electrodes and an (L+1)th row of electrodes among the K rows ofelectrodes, wherein L+2 is less than or equal to K, wherein the Lth rowof electrodes has a first polarity, and the (L+1)th row of electrodeshas a second polarity, and

(b) such that, subsequently, an (L+1)th electric field is appliedbetween the (L+1)th row of electrodes and an (L+2)th row of electrodes,wherein the (L+1)th row of electrodes has the first polarity, and the(L+2)th row of electrodes has the second polarity, and

d. repeating step c. as many times as desired with as many selections ofL as desired, such that L+2 is less than or equal to K.

In accordance with another aspect of the present invention, a method oftreating material with a treating agent is provided using pulsedelectrical fields provided by a waveform generator includes the stepsof:

obtaining an electrode assembly, which includes an array of electrodeswhich includes at least nine individual electrodes arrayed in a matrixof at least three parallel rows of electrodes and at least threeparallel columns of electrodes,

establishing electrically conductive pathways between the individualelectrodes and the waveform generator,

applying successive electric fields in the form of successive electricfield waveforms from the waveform generator to adjacent parallel rows ofelectrodes, wherein each successive electric field has the same firstdirection, and wherein polarities of rows of electrodes are reversedsuccessively during the applying of the successive electric fieldsbetween adjacent successive rows of electrodes in the first direction,and

applying successive electric fields in the form of successive electricfield waveforms from the waveform generator to adjacent parallel columnsof electrodes, wherein each successive electric field has the samesecond direction, and wherein polarities of columns of electrodes arereversed successively during the applying of the successive electricfields between adjacent successive columns of electrodes in the seconddirection, wherein the second direction is orthogonal to the firstdirection.

All individual electrodes in a row of electrodes can be permanentlyconnected together, and each row of electrodes can be connected to thearray switch. Alternatively, all electrodes can be individuallyconnected to the array switch.

The electrodes can be needle electrodes. The electric field intensitiesproduced by the electrodes can be 200 v/cm or greater. The electricpulse generator can produce one pulse per pair of rows of electrodesaddressed by the array switch. The electric pulse generator can producerectangular pulses from 1 microsecond to 1 second.

In accordance with another aspect of the present invention, an electrodeassembly is provided for connection to an array switch which isconnected to a pulse generator. The electrode assembly includes an arrayof electrodes which includes at least nine individual electrodes arrayedin a matrix of at least three parallel rows of electrodes and at leastthree parallel columns of electrodes, wherein each of the at least nineindividual electrodes is connected individually to the array switch.

With respect to the electrode assembly, each individual electrode isselectively connected to either a pulse generator anode, or a pulsegenerator cathode, or a neutral potential.

In accordance with another aspect of the present invention, acombination of an electrode assembly and an array switch is providedwhich is connected to a pulse generator. The combination includes anelectrode assembly which includes an array of electrodes which includesat least nine individual electrodes arrayed in a matrix of at leastthree parallel rows of electrodes and at least three parallel columns ofelectrodes, and an array switch is connected to the array of electrodes,wherein each of the at least nine individual electrodes is connectedindividually to the array switch.

Each individual electrode can selectively connected through the arrayswitch to either a pulse generator anode, or a pulse generator cathode,or a neutral potential.

In accordance with another aspect of the present invention, apparatus isprovided for the delivery of therapeutic compounds into biologicalcells. The apparatus includes a waveform generator. An array switch iselectrically connected to the waveform generator. An electrode assemblyis provided which includes an array of electrodes which includes atleast nine individual electrodes arrayed in a matrix of at least threeparallel rows of electrodes and at least three parallel columns ofelectrodes. The array of electrodes is electrically connected to thearray switch. Each of the at least nine individual electrodes isconnected individually to the array switch.

Each individual electrode can be selectively connected through the arrayswitch to either a waveform generator anode, or a waveform generatorcathode, or a neutral potential.

In accordance with another aspect of the present invention, apparatus isprovided for the delivery of therapeutic compounds into biological cellsin a treatment area. The apparatus includes a waveform generator. Anarray switch is electrically connected to the waveform generator. Anelectrode assembly is provided for placement upon the treatment area.The electrode assembly includes an array of electrodes which includes atleast nine individual electrodes arrayed in a matrix of at least threeparallel rows of electrodes and at least three parallel columns ofelectrodes. The array of electrodes is electrically connected to thearray switch. Each of the at least nine individual electrodes isconnected individually to the array switch, wherein each individualelectrode is selectively electrically connected through the array switchto either a waveform generator anode, or a waveform generator cathode,or a neutral potential.

Successive electric fields are applied to the treatment area in the formof successive electric field waveforms from the waveform generator toadjacent parallel rows of electrodes, wherein each successive electricfield has the same first direction, and wherein polarities of rows ofelectrodes are reversed successively during the applying of thesuccessive electric fields between adjacent successive rows ofelectrodes in the first direction.

In addition, successive electric fields are applied to the treatmentarea in the form of successive electric field waveforms from thewaveform generator to adjacent parallel columns of electrodes, whereineach successive electric field has the same second direction, andwherein polarities of columns of electrodes are reversed successivelyduring the applying of the successive electric fields between adjacentsuccessive columns of electrodes in the second direction. The seconddirection is orthogonal to the first direction.

The above brief description sets forth rather broadly the more importantfeatures of the present invention in order that the detailed descriptionthereof that follows may be better understood, and in order that thepresent contributions to the art may be better appreciated. There are,of course, additional features of the invention that will be describedhereinafter and which will be for the subject matter of the claimsappended hereto.

In this respect, before explaining preferred embodiments of theinvention in detail, it is understood that the invention is not limitedin its application to the details of the construction and to thearrangements of the components set forth in the following description orillustrated in the drawings. The invention is capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood, that the phraseology and terminologyemployed herein are for the purpose of description and should not beregarded as limiting.

As such, those skilled in the art will appreciate that the conception,upon which disclosure is based, may readily be utilized as a basis fordesigning other structures, methods, and systems for carrying out theseveral purposes of the present invention. It is important, therefore,that the claims be regarded as including such equivalent constructionsinsofar as they do not depart from the spirit and scope of the presentinvention.

In view of the above, it is an object of the present invention is toprovide a new and improved method of treating biological materials withtranslating electrical fields and electrode polarity reversal whichprovides an electroporation method in which the benefits of usingunipolar pulses are obtained without incurring the disadvantages ofunipolar pulses.

Still another object of the present invention is to provide a new andimproved method of treating biological materials with translatingelectrical fields and electrode polarity reversal that provides anelectroporation method in which the benefits of using bipolar pulses areobtained without incurring the disadvantages of the bipolar pulses.

Yet another object of the present invention is to provide a new andimproved method of treating biological materials with translatingelectrical fields and electrode polarity reversal which provides amethod of treating biological materials with electrical fields andtreating agents which employs unipolar pulses but which has minimaldeleterious electrolytic effects at the electrodes.

Even another object of the present invention is to provide a new andimproved method of treating biological materials with translatingelectrical fields and electrode polarity reversal that provides a methodof treating biological materials with electrical fields and treatingagents which employs unipolar pulses and retains good electrophoresisproperties for good cell uptake of treating agents.

Still a further object of the present invention is to provide a new andimproved method of treating biological materials with translatingelectrical fields and electrode polarity reversal which provides amethod of treating biological materials with electrical fields andtreating agents which employs unipolar pulses, but which also employselectrode polarity reversal.

Yet another object of the present invention is to provide a new andimproved method of treating biological materials with translatingelectrical fields and electrode polarity reversal that provides a methodof treating biological materials with electrical fields and treatingagents which employs electrode polarity reversal without employingbipolar pulses.

Additional advantages and the specific objects attained by its uses,reference should be had to the accompanying drawings and descriptivematter in which there are illustrated preferred embodiments of theinvention.

BRIEF DESCRIPTION OF DRAWINGS

The invention will be better understood and the above objects as well asobjects other than those set forth above will become more apparent aftera study of the following detailed description thereof. Such descriptionmakes reference to the annexed drawing wherein:

FIG. 1 illustrates a schematic diagram of a system used to produce theunidirectional, uniform electric fields.

FIG. 2 illustrates a schematic diagram of a specific arrangement ofpolarities for an array of electrodes as determined by a specificarrangement of switches in the array switch.

FIG. 3A schematically illustrates electric fields using two needles perrow of electrodes, wherein, in the left side of FIG. 3A, two rows ofelectrodes are spaced from each other by 4 mm, and wherein, in the rightside of FIG. 3A, two rows of electrodes are spaced from each other by 6mm.

FIG. 3B schematically illustrates electric fields using six needles perrow of electrodes, wherein, in the left side of FIG. 3B, two rows ofelectrodes are spaced from each other by 4 mm, and wherein, in the rightside of FIG. 3B, two rows of electrodes are spaced from each other by 6mm.

FIG. 4A is similar to FIG. 3B, left side, showing the electric field fora row of six anodes diametrically opposite a row of six cathodes.

FIG. 4B illustrates electric fields between a top row of four anodes, aparallel middle row of five cathodes, and a parallel bottom row of fouranodes, wherein electrodes in each row of electrodes are equidistantfrom the nearest electrodes in the adjacent row or rows of electrodes.

FIGS. 5A-5D schematically illustrate the progressive, unidirectionalmovement of the electric field vector through sequentially selected rowsof electrodes, accompanied by polarity of reversal of the rows ofelectrodes.

FIG. 6 schematically illustrates the unidirectional movement of afirst-direction progressing electric field vector through horizontalrows of electrodes in a first treatment area.

FIG. 7 schematically illustrates the unidirectional movement of asecond-direction progressing electric field vector through verticalcolumns of electrodes in a second treatment area, wherein thesecond-direction progressing electric field vector is orthogonal to thefirst-direction progressing electric field vector.

MODES FOR CARRYING OUT THE INVENTION

The treatment method uses the system illustrated in FIG. 1. There is apulse generator 12. A personal computer 13 is interfaced to the pulsegenerator 12. A RS-232 interface 15 can be used to interface thepersonal computer 13 to the pulse generator 12. An array switch 14 isconnected to the pulse generator anode 16 and pulse generator cathode18. The array switch 14 is also connected to a neutral or control 17.The array switch 14 is also connected either to each row of electrodesor to each electrode individually in the array of electrodes. One suchpulse generator is the Cyto Pulse Sciences, Inc. PA-4000, PulseAgile,generator. One such array switch is the Cyto Pulse Sciences, Inc. PA-201Programmable Pulse Switch. It is noted that the PA-201 ProgrammablePulse Switch is capable of being connected to up to thirty twoelectrodes or thirty-two rows of electrodes. As shown in FIG. 2. thePA-201 can connect the anode 16 or the cathode 18 of the pulse generatorto any row of the electrode array, if the rows are permanently wiredtogether, or to any electrode in the array if the rows of electrodes arenot permanently wired together.

A wide variety of electrodes can be employed. For example, theelectrodes can be solid needles, hollow needles, coated needles,uncoated needles, and porous needles.

To more fully appreciate the progressive wave electrode method of theinvention, some considerations of electric field in relation to rows ofin vivo electrodes will first be discussed.

If an electrode array consists of two active parallel rows, and each rowconsists of two electrodes, and one row is connected to the anode, andone row is connected to the cathode, then the electric field produced ispresented in FIG. 3A. The electric field calculation is shown as fieldlines 40 and 42. Reference is made to a perfect parallel plate with thesame outside dimension as the row length and the same spacing as the rowspacing. As the spacing between the electrodes in a specific rowincreases, or as the spacing between the electrode rows increases, or asthe needle diameter decreases, the electric field uniformity isdegraded.

FIG. 3B and FIG. 4A show the electric field calculation for a 6 needleper row electrode. The electric field calculation is shown as fieldlines 44 and 46. The conclusion of an unpublished internal Cyto PulseStudy dated October 1999 was that a nearly uniform electric field(within 20% of that produced by an equivalent parallel plate) resultsif:

1. The spacing distance between the electrodes rows 36 is at least 3times greater than the lateral distance between electrodes in a row 34.

2. The lateral length 38 of an electrode row is at least 2.5 times thespacing distance between the electrodes rows 36.

3. The diameter of the needle electrode is about 0.2 times the spacingdistance between the electrodes rows 36.

The electric field produced using just one or two needles per row (suchas shown in FIG. 3A) at best can produce only a very narrow uniformelectric field.

As shown in FIG. 4B, the equilateral triangle in which one row has oneelectrode and the second row has two electrodes of opposite polarity hasa complex electric field 48 which is not uniform and does not have anelectric field vector which is unidirectional (See U.S. Pat. No.5,873,849). More specifically, FIG. 4B shows the field pattern of athree row equilateral. As shown the equilateral has very limited treatvolume coverage and the electric field vectors point in two differentdirections.

To increase the treatment volume the spacing distance between theelectrodes rows 36 can be increased. As shown this reduces theuniformity of the electric field at the edges. As the distance increasesthe uniformity will go to zero. A larger spacing distance between theelectrodes rows 36 also requires the increase in voltage to maintain thesame electric field intensity in the middle.

Instead of increasing the row spacing to increase the treatment volume,additional rows can be added. If all rows are connected to the pulsegenerator simultaneously then the polarity of each row must alternate.This causes three problems. The first problem is that the electric fielddirection changes 180 degrees from one row to the next. The secondproblem is heating. Adding one row is effectively putting anotherresistor in parallel thus lowering the internal impedance of theelectrode when it is inserted in a conductive media such as livingtissue or an aqueous solution. The third problem is that by having morethan two row active means, more current from each row is required.

In an unpublished study by Cyto Pulse Sciences dated December 1999 andanother dated June 2000 the effect of connecting more than two rowssimultaneously was determined. The parameters of the electrodes used areset forth in Table 1 as follows: TABLE 1 Value Value Parameter UnitsDecember 99 June 00 Needle radius mm 0.082 0.15 Needle length mm 2.8 5.0Array length mm 8.3 12.2 Space between needles mm 3 5 Number of needles9 8 Number of rows 3 8

In the December study three rows were used and connected as shown inTable 2 as follows: TABLE 2 Run Row 1 Row 2 Row 3 1 + + + + + + − − − −− − 2 + + + + + + − − − − − − 5 + + + + + + − − − − − − + + + + + +

The needle array was placed in three homogeneous aqueous solutions andthe following resistances were measured, as shown in Table 3. TABLE 3Resistivity Aqueous Run 1 Run 2 Run 5 Run 5 solution Ohm-cm Pulse V/IPulse V/I Pulse V/I Ave Run 1 & 2 63 57.5 56.8 36.4 1.57 120 100.0 100.064.9 1.54 240 212.8 212.8 142.9 1.49

Adding the third row did not reduce the impedance of the electrode byhalf. This indicated that less current is flowing and thus the electricfield is less.

In the June study up to eight electrodes were used in an aqueoussolution and in beefsteak. Results of the June study are shown in Table4. Again the impedance of the array did not decrease as the elementaryassumption of adding another similar resistor. Thus as more rows areadded the electric field intensity is less than predicted. TABLE 4Number Of Rows Pulse V/I Aqueous Pulse V/I Beefsteak 2 29.4 106.7 3 20.570.6 4 13.7 48.0 5 10.7 39.8 6 8.90 32.9 7 7.36 28.1 8 6.09 23.7

The pulsing configuration for each row of a multi-parallel row electrodewith only one pair of rows of electrodes active at a time is shown inFIGS. 5A through 5D.

However, before discussing FIGS. 5A through 5D is detail, attention isfirst directed to FIG. 2. As shown in FIG. 2, in the array switch 14,each electrode can be connected by a selected switch 19 to either ananode (+) potential, a cathode (−) potential, or a neutral potential.For the specific selections illustrated in FIG. 2, electrode 1 (orelectrode row 1) is connected to the cathode potential. Electrode 2 (orelectrode row 2) is connected to the anode potential. Electrodes 3-8 (orelectrode rows 3-8) are connected to the neutral potential.

Turning to the discussion of FIGS. 5A through 5D, the array switch 14selections in FIG. 2 correspond to the selections for FIG. 5A.

Subsequently and not illustrated in FIG. 2, but illustrated in FIG. 5B,for electrode rows 1-5, electrode row 1 is connected to the neutralpotential. Electrode row 2 is connected to the cathode potential.Electrode row 3 is connected to the anode potential. Electrode rows 4and 5 are connected to the neutral potential.

Further, as illustrated in FIG. 5C, electrodes rows 1 and 2 areconnected to the neutral potential. Electrode row 3 is connected to thecathode potential. Electrode row 4 is connected to the anode potential.Electrode row is connected to the neutral potential.

Still further, as illustrated in FIG. 5D, electrode rows 1-3 areconnected to the neutral potential. Electrode row 4 is connected to thecathode potential. Electrode row 5 is connected to the anode potential.

Clearly, as illustrated in FIGS. 5A through 5D, the electric fieldvector 20 progressively moves unidirectionally. Moreover, the electricfield is uniform at each incremental position in the electric fieldprogression, such as through FIGS. 5A through 5D.

Furthermore, polarity reversals occur as the uniform electric fieldprogresses unidirectionally. More specifically in FIG. 5A, electrode row2 is connected to the anode potential. In FIG. 5B, electrode row 2 isconnected to the cathode potential.

In FIG. 5B, electrode row 3 is connected to the anode potential. In FIG.5C, electrode row 3 is connected to the cathode potential.

In FIG. 5C, electrode row 4 is connected to the anode potential. In FIG.5D, electrode row 4 is connected to the cathode potential.

It is understood that for electrode rows 1-5, the respective electrodesin the respective rows can be wired together. Alternatively, therespective electrodes in the respective rows of electrodes can beselected simultaneously by the array switch 14.

In the example in FIGS. 5A through 5D, a five elements by five elementselectrode is used. That is, 25 electrodes are arrayed in a matrix having5 rows and 5 columns. In general, an electrode array used with thepresent invention can be in a matrix array having K rows and M columns.

The Table 5 below provides the values of various parameters as afunction of distance between electrodes assuming the electrode areneedles. TABLE 5 Parameter Value Space Between 2 mm 3 mm 4 mm 5 mmNeedle Centers Coverage 6 × 8 mm 9 × 12 mm 12 × 16 mm 15 × 20 mm Time to0.5 0.5 0.5 0.5 complete one seconds seconds seconds seconds wave forPulse interval of 0.125 seconds Pulse Amplitude 240 volts 360 volts 480volts 600 volts for 1200 v/cm

With respect to the “coverage”, in FIG. 6 and FIG. 7, the actual areatreated is the area inside the 5×5 matrix array of electrodes. Also,with respect to FIG. 6 and FIG. 7, each of the twenty-five electrodes inthe 5×5 matrix array of electrodes is connected to the array switch 14individually.

More specifically with respect to FIG. 6, the electrodes are selected bythe array switch 14 so that groups of horizontal rows of electrodes 24are selected. In this respect, a first direction progressing electricfield vector 22 is oriented in a vertical direction in FIG. 6. Theactual area for ion minimization is first ion minimization area 26 whichis less than the area treated by the electric field. In this respect,the first-direction progressing electric field vector 22 is longer thanthe first ion minimization area 26 in the vertical direction.

More specifically with respect to FIG. 7, the electrodes are selected bythe array switch 14 so that groups of vertical columns of electrodes 30are selected. In this respect, a second-direction progressing electricfield vector 28 is oriented in a horizontal direction in FIG. 7. Theactual area for ion minimization is second ion minimization area 32which is less than the area treated by the electric field. In thisrespect, the second-direction progressing electric field vector 28 islonger than the second ion minimization area 32 in the horizontaldirection.

A treatment regimen can be provided so that a treatment area is treatedby (a) a first treatment by a progressive sequence of uniform electricfields advancing through the treatment area in a first direction,accompanied by polarity reversals of electrodes, such as shown in FIG.6, which is then followed by (b) a second treatment by a progressivesequence of uniform electric fields advancing through the treatment areain a second direction, which is orthogonal to the first direction,accompanied by polarity reversals of electrodes, such as shown in FIG.7.

It is apparent from the above that the present invention accomplishesall of the objects set forth, which include providing a new and improvedmethod of treating biological materials with translating electricalfields and electrode polarity reversal which may advantageously be usedto provide an electroporation method in which the benefits of usingunipolar pulses are obtained without incurring the disadvantages ofunipolar pulses. With the invention, a method of treating biologicalmaterials with translating electrical fields and electrode polarityreversal, provides an electroporation method in which the benefits ofusing bipolar pulses are obtained without incurring the disadvantages ofthe bipolar pulses. With the invention, a method of treating biologicalmaterials with translating electrical fields and electrode polarityreversal provides a method of treating biological materials withelectrical fields and treating agents which employs unipolar pulses butwhich has minimal deleterious electrolytic effects at the electrodes.With the invention, a method of treating biological materials withtranslating electrical fields and electrode polarity reversal provides amethod of treating biological materials with electrical fields andtreating agents which employs unipolar pulses and retains goodelectrophoresis properties for good cell uptake of treating agents. Withthe invention, a method of treating biological materials withtranslating electrical fields and electrode polarity reversal isprovided which provides a method of treating biological materials withelectrical fields and treating agents which employs unipolar pulses, butwhich also employs electrode, polarity reversal. With the invention, amethod of treating biological materials with translating electricalfields and electrode polarity reversal is provided which provides amethod of treating biological materials with electrical fields, andtreating agents which employs electrode polarity reversal withoutemploying bipolar pulses.

1. A method of treating material with a treating agent using pulsed electrical fields provided by a waveform generator, comprising the steps of: obtaining an electrode assembly which includes three or more parallel rows of individual electrodes, establishing electrically conductive pathways between the electrodes and the waveform generator, applying successive electric fields in the form of successive electric field waveforms from the waveform generator to adjacent rows of electrodes, wherein each successive electric field has the same direction, and wherein polarities of rows of electrodes are reversed successively during the applying of the successive electric fields between adjacent successive rows of electrodes.
 2. A method of treating material with an agent using pulsed electrical fields provided by a waveform generator, comprising the steps of: a. obtaining an electrode assembly which includes K rows of electrodes, where K is at least three, wherein each successive row of electrodes is spaced apart from a preceding row of electrodes, b. establishing electrically conductive pathways between the K rows of electrodes and the waveform generator, and c. providing successive electric fields in the form of successive electric field waveforms from the waveform generator to the K rows of electrodes, wherein each electric field has the same direction, (a) such that an Lth electric field is applied between a selected Lth row of electrodes and an (L+1)th row of electrodes among the K rows of electrodes, wherein L+2 is less than or equal to K, wherein the Lth row of electrodes has a first polarity, and the (L+1)th row of electrodes has a second polarity, and (b) such that, subsequently, an (L+1)th electric field is applied between the (L+1)th row of electrodes and an (L+2)th row of electrodes, wherein the (L+1)th row of electrodes has the first polarity, and the (L+2)th row of electrodes has the second polarity, and d. repeating step c. as many times as desired with as many selections of L as desired, such that L+2 is less than or equal to K. 3-6. (canceled)
 7. The method of claim 2 wherein the pulsed electric field waveforms are from electrical pulses which are in a sequence of at least three non-sinusoidal electrical pulses, having field strengths equal to or greater than 100 V/cm, to the material, wherein the sequence of at least three non-sinusoidal electrical pulses has one, two, or three of the following characteristics: (1) at least two of the at least three pulses differ from each other in pulse amplitude; (2) at least two of the at least three pulses differ from each other in pulse width; and (3) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses.
 8. The method of claim 2 wherein the first polarity is positive and the second polarity is negative.
 9. The method of claim 2 wherein the first polarity is negative and the second polarity is positive.
 10. The method of claim 2 wherein successive electric fields are applied from the first and second rows of electrodes to the Kth row of electrodes.
 11. The method of claim 2 wherein successive electrical fields are applied from the Kth row of electrodes and (K−1)th row of electrodes to the first row of electrodes.
 12. The method of claim 2 wherein the material being treated includes biological material. 13-18. (canceled)
 19. The method of claim 2 wherein the treating agent includes molecules of electrode releasable tissue treating agent on the electrodes, and wherein the molecules of the electrode releasable tissue treating agent are released from the electrodes by contacting the electrodes with a solvent.
 20. A method for immunotherapy, comprising the steps of: a. obtaining an electrode assembly which includes K rows of electrodes, where K is at least three, wherein each successive row of electrodes is spaced apart from a preceding row of electrodes, wherein each electrode is statically-coated with an immuno-stimulating material, b. establishing electrically conductive pathways between the K rows of electrodes and a waveform generator, c. inserting the statically-coated electrodes into a tissue to be treated, d. releasing the immuno-stimulating material from the electrodes, e. providing successive electric fields in the form of successive electric field waveforms from the waveform generator to the K rows of electrodes, such that the released immuno-stimulating material is driven into cells in the tissue, wherein each electric field has the same direction, (a) such that an Lth electric field is applied between a selected Lth row of electrodes and an (L+1)th row of electrodes among the K rows of electrodes, wherein L+2 is less than or equal to K, wherein the Lth row of electrodes has a first polarity, and the (L+1)th row of electrodes has a second polarity, and (b) such that, subsequently, an (L+1)th electric field is applied between the (L+1)th row of electrodes and an (L+2)th row of electrodes, wherein the (L+1)th row of electrodes has the first polarity, and the (L+2)th row of electrodes has the second polarity, and f. repeating step e. as many times as desired with as many selections of L as desired, such that L+2 is less than or equal to K. 21-46. (canceled)
 47. A method of treating material with a treating agent using pulsed electrical fields provided by a waveform generator, comprising the steps of: obtaining an electrode assembly which includes an array of electrodes which includes at least nine individual electrodes arrayed in a matrix of at least three parallel rows of electrodes and at least three parallel columns of electrodes, establishing electrically conductive pathways between the individual electrodes and the waveform generator, applying successive electric fields in the form of successive electric field waveforms from the waveform generator to adjacent parallel rows of electrodes, wherein each successive electric field has the same first direction, and wherein polarities of rows of electrodes are reversed successively during the applying of the successive electric fields between adjacent successive rows of electrodes in the first direction, and applying successive electric fields in the form of successive electric field waveforms from the waveform generator to adjacent parallel columns of electrodes, wherein each successive electric field has the same second direction, and wherein polarities of columns of electrodes are reversed successively during the applying of the successive electric fields between adjacent successive columns of electrodes in the second direction, wherein the second direction is orthogonal to the first direction.
 48. An apparatus for the delivery of therapeutic compounds in vivo or in vitro into living cells comprising: an array of electrodes consisting of three or more parallel rows of electrodes with more than three electrodes per row with the electrodes in each row opposing the electrodes in adjacent rows of electrodes, an electrical pulse voltage generating means with an anode and a cathode, and an array switching means which connects the anode of the pulse voltage generator to a row of electrodes and the cathode of the pulse generator to an adjacent row of opposing electrodes, wherein said array switching means if operated for successively selecting a succeeding pair of rows of electrodes, such that only one row of the next pair of rows of electrodes must have been connected during the previous pair connection, and such that the polarity of the common row of electrodes connected be opposite for the next connected pair of rows of electrodes, and successively connecting the next adjacent rows of electrodes in the same manner until all rows of electrodes have been connected to said pulse generator.
 49. The apparatus of claim 48 wherein all individual electrodes in a row of electrodes are permanently connected and wherein each rows of electrodes is connected to the array switch.
 50. (canceled)
 51. The apparatus of claim 48 wherein said electrodes are needle electrodes.
 52. The apparatus of claim 48 wherein electric field intensities produced by the electrodes are 200 v/cm or greater.
 53. The apparatus of claim 48 wherein said electric pulse generator produces one pulse per pair of rows of electrodes addressed by said array switch. 54-57. (canceled)
 58. An apparatus for the delivery of therapeutic compounds in vivo or in vitro into living cells comprising: an array of electrodes consisting of three or more parallel rows of electrodes with more than three electrodes per row with the electrodes in each row opposing; and an electrical pulse voltage generating means with an anode and a cathode, and an array switching means which connects the anode of the pulse voltage generator to a row of electrodes and the cathode of the pulse generator to an adjacent row of opposing electrodes, and successively selecting the next pair such that only one row of the next pair must have been connected during the previous pair connection and the polarity of the common row connected but be opposite for the next connected pair and successively connecting the next adjacent row in the same manner until all rows have been connected in one direction and then connecting rows of the array in an orthogonal direction and repeating the connection and pulsing process as above. 59-65. (canceled) 